DE102005026133B4 - Method for determining the antioxidant activity of substances and mixtures of substances - Google Patents

Abstract

Method for determining the antioxidant activity of substances or substance mixtures, in particular raw materials or finished products, by measuring the reduction of at least one test radical with at least one antioxidant substance contained in the substances or substance mixtures,
marked by
a) at least one measurement of a temporal dependence of a signal intensity I as a measure of the number of free test radicals N at least two different weights w _n (n = 1, 2 ..) of the antioxidant substance to be tested,
b) Generation of at least one first data set for determining a first, dynamic parameter in the form of the reaction time t _r from the signal intensities I measured for at least two different times t _m (m = 1, 2) for the respective initial weights w _n (n = 1, 2, ...) of the antioxidant substance to be tested [I (t) = I _wn (t)],
c) generating at least one second data set for determining a second, static parameter in the form of the characteristic weight w _c from the functional relationship of the measured signal intensities I of the ...

Description

The The invention relates to a method according to the preamble of the claim 1.

oxidative Processes that take place in living organisms result in the Formation of highly reactive free radicals. These radicals influence and accelerate the aging process of organisms and impair health. So probably radicals are the cause of arteriosclerosis and cancer.

It has long been known that certain antioxidant substances, the ability possess to interrupt the radical reactions caused by radicals and disable radicals.

In biological research, medicine, pharmacy and food technology is becoming increasingly the antioxidant potential of naturally occurring Antioxidants studied. A rich source of antioxidant active substances represent plants, especially vegetables and medicinal herbs.

to assessment The antioxidant effectiveness of substances becomes a variety used by analytical methods and measurement methods. As a general rule The measurement methods are based on the ability of antioxidants, disable free radicals.

From the DE 43 28 639 A1 a method for the determination of the antioxidant potential in the skin is known. In this process, a free radical bearing nitroxide is applied to a skin sample to be examined. Subsequently, the magnetic moments of the free radicals are measured under specific conditions by means of electron spin resonance spectroscopy and a ratio is formed between two measured values, one of which was obtained by performing an intermediate step during which oxidative stress was applied to the skin and the other without this intermediate step.

From the DE 198 30 004 A1 a method for the determination of a radical protection factor is known in which the radical reduction is determined by a test substance by means of normalized subtraction and is obtained by subsequent normalization to the amount of the test substance used, the radical protection factor. This radical protection factor is a time-independent value.

From the WO 02/061422 A1 For example, a method for determining the antioxidant activity of a cell is known. The measured fluorescence of a fluorescent substance is converted into the concentration of the fluorescent substance. The concentration then serves as a measure of the antioxidant activity of the cell.

The Previously known methods use organic radicals, either while The measuring process itself can be generated or as a semi-stable highly reactive free radicals are supplied to the substance to be examined (Schlesier et al., Assessment of antioxidant activity by using different in vitro methods, Free Radical Research, 2002, 36: 177-187). The Procedures differ essentially by the process of Radical formation or radical reduction.

Three Test methods (TEAC I, TRAP, PCL) measure the delay of the Radical formation (oxidation) and determine the delay time (Lag phase) as a parameter for the characterization of the antioxidant Activity. she determine the delay (delay) of radical generation and the ability to deactivate radicals to reduce.

So is measured in the TEAC I assay (Trolox equivalent antioxidant capacity I-assay) the oxidation of ABTS by H2O2 and metmyoglobin to ABTS · -Radikals in the presence of the antioxidant substance photometrically 734 nm measured. Dependent on from the concentration and reactivity of the radical scavenger used delayed the oxidation of ABTS and the antioxidant potential of the substances by determining the Lag phase determined.

in the TRAP Assay (Total Radical-Trapping Antioxidant Parameter) will be the Fluorescence of R-phycoerythrin quenched with ABAP as a radical generator. By adding antioxidants the decomposition of R-phycoerythtrin is inhibited and the antioxidant Potential determined by measuring the lag phase.

Both assays are thus based on measuring the delay of free radical oxidation by addition of antioxidants. The lag phase serves as a parameter for the antioxidant capacity of a sub substance. The delay of radical formation as well as the radical scavenging ability can thus be estimated.

in the In contrast, the other methods (TEAC II, TEAC III, DPPH, DMPD) the ability the antioxidants to directly reduce a radical. This is the decrease in the absorption of a radical in the presence of an antioxidant effective substance.

The TEAC assay II (Trolox equivalent antioxidant capacity II-assay) is based on the formation of ABTS ^{· -} radical by filtering an ABTS solution by MnO ₂ powder. The solution of the ABTS.RTM ^. Radical prepared in the filtering process is mixed with the antioxidant and the antioxidant activity is measured by measuring the absorbance decrease at 734 nm using the equation [% antioxidant activity = ((E _ABTS.E -E _standard ) / E _{ABTS. -} ) × 100].

At the DPPH Assay (Diphenyl Picryl Hydrazyl) is the free 2,2-diphenyl-1-picrylhydrazyl radical reduces the antioxidant. The antioxidant effect of the measured Substance is determined by measuring the altered absorption of the radical determined. The DPPH assay will be used to evaluate the antioxidant activity of food as well as more recently Time to Quantify Antioxidants in Complex Biological Systems used. The DPPH method is applicable to liquid and solid systems and is not limited on the determination of the antioxidant activity of a specific substance, but to the total activity a sample.

Usually The DPPH assay is performed by measuring the extinction coefficient performed in a VIS spectrophotometer, which with a strong Absorption at 517 nm is associated. The free radical has one Violet color, which changes to yellowish, as soon as the free An electron of radical containing a hydrogen atom from a radical scavenger Formation of DPPH-H reacted. The resulting discoloration is in a stoichiometric relationship to the number of trapped electrons.

A more accurate method of measuring antioxidant activity in particular of colored and dull substance solutions represents the electron spin resonance (ESR) spectroscopy at which directly measures a signal of the free radical electron becomes.

antioxidants in foods are usually water-soluble or fat-soluble and react in different ways with free radicals. The reaction time the antioxidants with radicals varies greatly. That's how the duration can be Assays are in the minute or hour range.

In most of the assays described in the literature the free radical with the entire sample until a steady state the reaction (steady state) is reached. This process can, for. B. in the case of the DPPH method up to 6 hours depending from the molecular structure of the antioxidant substance and the used solvents to last; to endure, to continue.

Therefore is used in most publications as the endpoint of the determination of antioxidant activity considering the value of an unknown antioxidant a sample where the value of the initial signal of a radical is reduced by 50% Has.

This Approach, however, has a number of disadvantages. First, the lead different in vitro test systems to different results, making a simple comparison of the antioxidant activity of substances Certainly not possible with different assays.

To the another is a long measurement time of several unknown samples necessary antioxidant substance. A minimum of three measurements each with different weights until reaching the steady state state must be done become a functional reduction curve of the radical signal to represent as a function of the weight weighed. The from these measurements obtained values for the determination of antioxidant activity how the value of Antiradical Power (ARP) is just information about the antioxidant capacity the substance, but not over the reactivity, the above the reduction kinetics will be described.

Because aggressive free radicals, due to their high reactivity, are usually very short-lived, their deactivation requires reactive virulent antioxidants. It is therefore not appropriate to determine the antioxidant activity or capacity of a substance over a long period of up to 6 hours. This is only a general statement about the reduction capacity of antioxidant substances gegenü Struck by free radicals, which makes no statements about their effectiveness.

Of the Information content of the procedures TEAC I, TRAP and PCL is higher because he additionally information about the reactivity contains the antioxidant substance, represented by the lag phase. The quantification with regard to However, the reduced amount of free test radicals is far and away more difficult because the test radical is generated immediately during the measurement process and immediately reduced again by the antioxidant. The process of Radical generation is affected by many parameters such as the Solvent, the reactants, the pH and the temperature, which cause strong fluctuations of the results. An exact reproducible Measurement is hardly possible. The mentioned methods mostly allow only comparative measurements between similar products.

From the procedures TEAC II, TEAC III, DPPH and DMPD, which the capacity of one antioxidant substance, a certain amount of test radicals to disable or reduce measure is the DPPH method the simplest and most uncomplicated in the application. It delivers the most stable results and has a wide application. adversely in the procedure is its limited expressiveness, which no information about the activity the time reduction kinetics of the antioxidant substance mediates.

Of the The invention is therefore based on the problem of providing a measuring method, with which parameters are determined, the statements about the Reactivity the temporal radical reduction and the capacitive reduction capacity of allow antioxidant substance.

These Task is achieved by a method with the features of claim 1 solved.

Thereafter, the inventive method for determining the antioxidant activity of substances or mixtures, in particular of raw materials and finished products, by measuring the reduction of at least one test radical with at least one antioxidant substance, characterized in that at least one measurement of a temporal dependence of a signal intensity I as a measure of the number of free test radicals N at least two different weights w _n (n = 1, 2, ...) of the antioxidant substance to be tested is carried out.

From the signal intensities I measured for at least two different times t _m (m = 1, 2) for the respective initial weights w _n (n = 1, 2,...) _Of the antioxidative substance to be tested [I (t) = I _wn ( t)], at least one first data set for determining a first, dynamic parameter in the form of the reaction time t _{r is} generated.

Following this, at least one second data set from the functional relationship of the measured signal intensities I of the respective initial weights w _n (n = 1, 2,...) Of the antioxidant substance to be tested at time t _r , [I _{(t = tr)} = f (w)], with which a second, static parameter in the form of the characteristic weight w _{c is} determined.

To determine a third, two-dimensional parameter in the form of the Antioxidative Power AP, at least a third data set is generated. The Antioxidant Power AP parameter is a quantitative time-dependent measure of antioxidant activity as a two-dimensional ratio AP (t, w) ~ N / (t r · w c ). described.

With the value of the Antioxidative Power AP is defined as a parameter the ability an antioxidant substance, at a certain time interval dependent on a certain number of the amount of antioxidants used of radicals. In the parameter AP are thus two Properties of the antioxidant substance integrated: the antioxidant capacity as a static part and the reactivity as a dynamic part. The Differentiation of the AP into these two shares allows one more detailed and comprehensive description of the antioxidant properties a substance. The consideration the kinetic behavior of the reduction process of an antioxidant Substance together with the use of a stable and established Test method represents a new quality compared to previously known Methods dar.

• a) Auswertung mindestens zweier unterschiedlicher Einwaagen w_n (n = 1, 2, ...) der antioxidativen Substanz und Auswertung der Veränderung der Signalintensität des Testradikals I_wn(t) (w_n = w₁, w₂, ...) (Einheit Arbitrary Units) für die mindestens zwei Einwaagen zu mindestens zwei verschiedenen Zeitpunkten t_m (m = 1, 2, ...),
• b) Normierung der jeweiligen gemessenen Signalintensitäten I_wn(t) = I(t) auf ihren jeweiligen Ausgangswert I_wn(t = 0) = I₀, Erzeugung eines Datensatzes mit n (n = 1, 2, ...) vergleichbaren normierten Reduktionskurven mittels Regression der durch die gemeinsame Einwaage w_n zusammengehörenden normierten Signalintensitäten mit einer monoexponentiellen Funktion i_wn(t) und Bestimmung des funktionellen Zusammenhangs i_wn(t)/I₀ = f(t) gemäß
  
  wobei I_0n der Signalintensität entspricht, die am Sättigungspunkt der Radikalreduktion für die jeweilige Einwaage w_n erreicht ist, und Bestimmung der jeweiligen Reaktionskonstante des Reduktionsprozesses k_wn (w_n = w₁, w₂, w₃, ...) mit der Maßeinheit min^–1 für die jeweilige Einwaage, und
• c) Bestimmung der Reaktionszeit t_rn (Einheit min) durch die auf I₀ normierten Gleichung trn = [–ln(1 – x)]/kwn (2).

The reaction time t _r as a parameter describing the dynamics and reactivity of the antioxidant substance is advantageously determined by means of the following, automatically proceeding steps:

• a) Evaluation of at least two different initial weights w _n (n = 1, 2,...) of the antioxidative substance and evaluation of the change in the signal intensity of the test radical I _wn (t) (w _n = w ₁ , w _{2 ,.} (Unit Arbitrary Units) for the at least two weights at least two different times t _m (m = 1, 2, ...),
• b) normalization of the respective measured signal intensities I _wn (t) = I (t) to their respective output value I _wn (t = 0) = I ₀ , generation of a data set with n (n = 1, 2, ...) comparable normalized Reduction curves by regression of the normalized signal intensities associated with the common weight w _n with a mono-exponential function i _wn (t) and determination of the functional relationship i _wn (t) / I ₀ = f (t) according to
  
  where I _0n corresponds to the signal intensity reached at the saturation point of the radical reduction for the respective weight w _n , and determination of the respective reaction _{constant of} the reduction process k _wn (w _n = w ₁ , w ₂ , w ₃ ,...) with the unit of measurement min ^-1 for the respective weight, and
• c) Determination of the reaction time t _rn (unit min) by the equation normalized to I ₀ t rn = [-Ln (1 - x)] / k wn (2).

The reaction time t _rn is advantageously determined by an automatic evaluation of the functional relationship I _rn (t _rn ) according to I rn (t rn ) = I 0n + (1 - x) (I 0 - I 0n ) = I 0n + (I 0 - I 0n ) exp - (k wn · t rn ) (3) With (1 - x) = exp - (k wn · t rn ) (4) generated.

The reaction time t _rn determines the period in which the normalized signal intensity i _wn (t = 0) / I ₀ = I ₀ / I ₀ = 1 of the test radical decreased by a factor of x, and is used to characterize the dynamics and reactivity of the radical scavenging capacity the antioxidant substance used.

The factor x is preferably an arbitrary number to be selected between 0... 1, which is constant for all weights w _n and describes the time t _rn = t _r , where t _{r is} an averaging of all experimentally determined t _rn values in which the signal intensity I ₀ has fallen by x times the total signal _decrease I _{0 -I 0n} of the total reduction _capacity due to the weight w _n .

The factor x can preferably take the value of x = 1 / e = 0.368, so that t rn = 0.462 / k wn (5) is.

Normally the reaction times are the same, so that t _r = t _r1 = t _r2 = t _rn . Inaccuracies in the regression, however, require an averaging of all experimentally determined t _rn values according to t r = Σt rn / n (6).

• a) Anordnung der für den Zeitpunkt t_rn = t_r ermittelten Signalamplituden I_rn(t_rn)/I₀ als Funktion ihrer zugeordneten Einwaagen w = w_n (n = 1, 2, ...), Erzeugung des funktionellen Zusammenhanges I_rn(t_rn)/I₀ = f(w_n), und Regression der sich aus dem funktionellen Zusammenhang ergebenen Funktionskurve durch eine monoexponentielle Funktion gemäß
  
• b) Bestimmung der Reaktionskonstante k_tr (Einheit mg^–1) aus der monoexponentiell angepassten Funktionskurve, und
• c) Bestimmung der charakteristischen Einwaage w_c (Einheit mg/ml) als Menge, die notwendig ist, das maximale mögliche Reduktionsvermögen von x = (x·I₀)/I₀ um den Faktor x von I₀ auf I_c zu vermindern, gemäß
  

To characterize the antioxidant capacity as a parameter that describes the total ability of a substance to reduce free radicals without regard to its reaction dynamics, is the Weighing mass w _c , which is referred to as a characteristic weight. The value of the characteristic weighing mass w _c is preferably determined by means of the following, automatically proceeding steps:

• a) Arrangement of the determined for the time t _rn = t _r signal amplitudes I _rn (t _rn ) / I ₀ as a function of their associated weights w = w _n (n = 1, 2, ...), generating the functional relationship I _rn (t _rn ) / I ₀ = f (w _n ), and regression of the function curve resulting from the functional relationship by a mono-exponential function according to
  
• b) Determination of the reaction constant k _tr (unit mg ^-1 ) from the mono-exponentially adjusted function curve, and
• c) determination of the characteristic weight w _c (unit mg / ml) as an amount which is necessary to reduce the maximum possible reducing power of x = (x * I ₀ ) / I ₀ by the factor x from I ₀ to I _c according to
  

Advantageously, w _{c is} determined by the automatic evaluation of the function I _c (w _c ) according to I c (w c ) = (1 - x) I 0 + (I 0 - (1 - x) I 0 ) (1 - x) = (1 - x) I 0 + (I 0 - (1 - x) I 0 ) exp - (k tr · w c ) (9) With (1 - x) = exp - (k tr · w c ) (10) certainly.

bestimmt wird.Preferably x assumes the value 1 / e, whereby the characteristic mass according to

is determined.

The parameter of the Antioxidative Power AP is given by the equation AP = (RA · N) / (t r · w c ) (12) where RA represents the characteristic radical degradation for the test radical.

The radical degradation determination record RA is according to the equation RA = 1 - (1 - x 2 ) = x 2 (13) RA, whereby RA by an automatic evaluation of the functional relationship I _c (RA) according to RA = I 0 / I 0 - I c / I 0 (14) With I c = (1 - x) I 0 + (I 0 - (1 - x) I 0 ) · (1 - x) = I 0 - x 2 I 0 and I c / I 0 = 1 - x 2 (15) is determined.

RA as a reduction factor is thus defined by the factor x, which can take any value useful in the context of the present invention. Preferably, RA has a value of RA = x ² = (1 / e) ² = 0.135.

N is the amount of reduced free radicals characterized by their electron spin. AP is preferred in the units [spins / min · mg] or [mol / l · min · mg].

With Advantage is the Antioxidant Power AP in the standard antioxidant unit Unit (AU), where 1 AU is the Antioxidant Power of a solution of Vitamin C at a concentration of 1 ppm corresponds.

The introduction of the standard unit AU allows a direct comparison of the determined antioxidative abilities of different substances and their relation to the Antioxidative Power AP of 1 ppm Vitamin C.

The Measurement of signal intensity I of the test radical is advantageously carried out by means of ESR spectroscopy. When Test radicals are preferably 2,2-diphenyl-1-picrylhydrazil (DPPH), 2,6-di-tert-butyl-alpha- (3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidenes) -p-tolyloxy (galvinoxyl), Fry's salt (solid salt of nitroso-disulfonic acid) and / or Nitroxyde used.

The signal intensity I of the test radical can also be colorimetrically in a UV / VIS Spectrometer using 2,2-diphenyl-1-picrylhydrazil (DPPH) can be measured as a test radical.

The Invention will be described below with reference to the figures in several embodiments explained in more detail. It demonstrate:

1 : a diagram in which the decrease of the normalized signal intensities i _w (t) / I _{0 of} a test radical as a function of time and due to three different weights w _n (n = 1, 2, 3) of an antioxidant substance is plotted;

2 FIG. ₂ : a second diagram in which the normalized signal intensities I _rn (w _n ) / I _{0 of} the test radical, recorded after a reaction time t _r , are fitted as a mono-exponential function i (w) / I ₀ = i _tr (w) / I ₀ weighed amounts w of the antioxidant substance is applied;

3a FIG. 3 is a third graph showing the antioxidant power AP of various types of tea tested according to their AU values (bar graph) and as a function of the respective reaction times t _r (line graph); and

3b FIG. 4: a fourth diagram showing the antioxidant power of various types of tea tested according to their AU values (bar chart) and as a function of the respective weighted weights w _c (line chart).

Exemplary embodiment 1: Determination of the reaction time t _r

The Measurement and calculation of individual parameters for determination The antioxidant Power AP is exemplified by green tea as radical scavengers shown.

For this purpose, initially three different amounts w _n of green tea (w ₁ = 0.01 mg / ml, w ₂ = 0.02 mg / ml, w ₃ = 0.03 mg / ml) are weighed. To measure the reaction time t _r , the initial weights are dissolved in a water / ethanol solution (50/50). 200 μl of this solution is mixed with 200 μl of a test radical solution of 0.2 mM DPPH in pure ethanol to give a DPPH final concentration of 0.1 mM in a water / ethanol solution (25/75). Immediately after mixing the substance solution with the DPPH solution, the ESR measurement is started in an ESR device from Magnettech (MS 200). At five different times (t ₁ = 1 min, t ₂ = 4 min, t ₃ = 10 min, t ₄ = 20 min and t ₅ = 30 min), the decrease in signal intensity is measured. The data resulting from the normalized signal intensities I _wn (t) / I ₀ for the respective weight are fitted with mono-exponential functions i _wn (t) / I ₀ (see 1 ). The resulting reaction _constants k _wn for each of the three initial _weights are k _w1 = 1.325 min ^-1 , k _w2 = 1.305 min ^-1 and k _w3 = 1.346 min ^-1 . The corresponding reaction times result according to equation (2) for the three initial weights t _r1 = 0.349 min, t _r2 = 0.354 min and t _r3 = 0.343 min, resulting in an average value for t _r according to equation (6) of 0.349 min. The mean for t _r represents the first parameter of the antioxidant power and determines the dynamics and reactivity of the radical scavenger process caused by the antioxidant ingredients of the green tea.

Embodiment 2: Determination of Reaction Capacity w _c

For the determination of the second parameter of the characteristic weight w _c , which characterizes the reaction capacity, a second diagram is necessary, in which the normalized signal intensities I _rn / I _{0 are represented} at time t _r as a function of the respective weight w _n of green tea ( please refer 2 ). A mono-exponential fit of these data according to equation (2) gives a reaction constant k _tr of 45.87 mg ^-1 . For the reaction capacity w _c, this results in accordance with equation (3) of 0.01007 mg of green tea.

embodiment 3: Determination of Antioxidant Power AP

mit
RA = 0.137;
N (DPPH-spins): 0.1 mM ^ 6.022·10¹⁶ spins/ml;
t_r = 0.349 min; und
w_c = 0.01007 mg,
woraus sich gemäß

ein AP Wert von 2.376·10¹⁸ spins/mg·min ergibt.This results in an AP value according to equation (12) for green tea

With
RA = 0.137;
N (DPPH spins): 0.1 mM ^ 6.022 x 10 ¹⁶ spins / ml;
t _r = 0.349 min; and
w _c = 0.01007 mg,
from which according to

an AP value of 2,376 x 10 ¹⁸ spins / mg.min.

embodiment 4: Introduction the standard unit Antioxidative Unit (AU)

Since the definition of the antioxidant Power AP by the unit [spins / mg.min] is too cumbersome for a uniform view and comparison of the antioxidant activities of the measured substances, the unit Antioxidative Unit (AU) is introduced. This unit is based on the antioxidant power of a 1 ppm vitamin C solution. The Antioxidative Power AP of a 1 ppm VitC solution is 2,495 · 10 ¹³ spins / mg · min and is defined as 1 Antioxidant Unit AU. Based on this, the calculated antioxidant power AP of the green tea of AP = 2,376 · 1018 [spins / mg · min] results in an AU value of 0.95229 · 10 ⁶ AU.

Using the method described, the values of the antioxidant Power AP were determined for five different teas. Table 1 compares the calculated AP values for the teas chamomile, green tea, black tea, herbal tea and rose hip hibiscus tea. Table 1 AP (spins / mg.min) AU t _r (min) w _c (mg / ml) chamomile 7.296 × 10 ¹⁶ 2923 0.634 0,178 Green tea 2,376 · 10 ¹⁸ 95229 0.349 0,010 Black tea 3,617 · 10 ¹⁷ 14499 0.745 0.0306 Herbal tea 2,484 · 10 ¹⁷ 9955 0.881 0.0377 Rosehip Hibiscus Tea 4.356 · 10 ¹⁶ 1746 2,249 .0842

Green tea has not only excellent antioxidant capacity but also very good reactivity, resulting in high value for the antioxidant Power AP. The AP of Green Tea is 6 times larger than Black Tea, a point commonly known. While green tea has antioxidant properties due to a short reaction time of t _r = 0.349 min, the rose hip hibiscus tea has a 6.44 times longer reactivation time t _r of 2.249 min. Thus, in order to obtain a signal intensity reduction of the DPPH radical by RA = (1 / 2e) ² , a characteristic weight of w _c = 0.0842 mg rosehip hibiscus tea must be used, for a total of 8.42- sometimes more than the necessary amount of green tea. The reactivity and reaction capacity of black tea and herbal tea differ only slightly.

With the exception of chamomile tea, the antioxidant power of the tested teas is determined equally by the reactivity and the reaction capacity. In contrast, chamomile tea has a high reactivity of 0.634 min, but the reaction capacity of 0.178 mg / ml is significantly higher than that of the other teas. From these values it can therefore be concluded that the concentration of the Antioxidant component in chamomile tea is many times lower than in other teas. The AP value thus gives information about the quality in the form of the time parameter of the reaction time t _r and the quantity in the form of the stationary parameter of the reaction capacity w _c .

1 shows the graphic determination of the reaction time t _r , which results from the functional relationship of the normalized signal intensities I / I ₀ = I _wn (t) / I ₀ of three weights w _n and the corresponding reaction times t of the test radical DPPH. The experimentally determined values are matched according to their identity (same weight w _n ) by means of a mono-exponential function i _wn (t) / I ₀ . The value t _rn represents for the respective function i _wn (t) / I ₀ the time after which the signal intensity of the DPPH radical is a value of 1 / e, the radical _reduction I _{0 -I 0n} determined by the respective weight w _n , has decreased. The resulting normalized intensity values for the test radical are I _r1 / I ₀ = 0.849, I _r2 / I ₀ = 0.793 and I _r3 / I ₀ = 0.713 s. The maximum possible signal reduction, which is characterized by the steady-state, depends on the amount w _{n of} the antioxidant substance used and is characterized by the normalized values I ₀₁ / I ₀ = 0.593 for w ₁ , I ₀₂ / I ₀ = 0.441 for w ₂ and I ₀₃ / I ₀ = 0.225 for w ₃ .

2 shows the normalized signal intensities I _rn / I ₀ for a 0.1 mM starting solution of the DPPH radical after corresponding reaction times t _rn , which are a mono-exponential function i _tr (w) / I _{0 of} the respective weights w with w = w _n (n = 3) of the green tea. The calculated value according to the embodiment of the characteristic weight (reaction capacity) w _{c of} the green tea is 0.01007 mg / ml. As can be seen from the diagram, the signal intensity I _{c of} the DPPH radical I _c remaining at this w _c value is _c = 0.863 · I ₀ . For the mono-exponential curve this results in a value of I _c / I ₀ = 0.863. This means that 0.01007 mg / ml green tea reduces the signal of the DPPH radical (0.1 mM) by 13.7%.

3a shows the correlation of the value of the Antioxidative Power AP of the tested teas with the reaction times t _r , while 3b represents the correlation of the AP value with the characteristic initial weights (reaction capacities) w _c determined for this purpose. The fact that in both figures, no clear inverse correlation between the AP value, and the values of _r and t w _c is clearly pointed out that the various types of tea containing different antioxidants with different degrees of free-radical scavenger properties. The different reaction times show that in addition to the amounts used, different antioxidants in the individual teas determine the AP value. Thus, the long reaction time of rosehip hibiscus tea indicates other effective antioxidants in this tea than green tea.

Claims (19)

Method for determining the antioxidant activity of substances or substance mixtures, in particular raw materials or finished products, by measuring the reduction of at least one test radical with at least one antioxidant substance contained in the substances or substance mixtures, characterized by a) at least one measurement of a temporal dependence of a signal intensity I as a Measure of the number of free test radicals N for at least two different weights w _n (n = 1, 2 ..) of the antioxidative substance to be tested. B) Generation of at least one first data set for determining a first, dynamic parameter in the form of the reaction time t _r from the signal intensities I measured for at least two different times t _m (m = 1, 2) for the respective weighing weights w _n (n = 1, 2,...) of the antioxidative substance to be tested [I (t) = I _wn ( t)], c) generating at least one second data set for determining a second, static parameter s in the form of the characteristic weight w _c from the functional relationship of the measured signal intensities I of the respective weights w _n (n = 1, 2,...) of the antioxidative substance to be tested at time t _r , [I _{(t = tr)} = f (w)], and d) generating at least a third data set for determining a third parameter in the form of the antioxidant power AP, which uses a two-dimensional quotient as a time-dependent, quantitative measure for evaluating the antioxidant activity of an antioxidant substance

is. A method according to claim 1, characterized in that the dynamic parameter of the reaction time t _{r is determined} by means of the following automatically proceeding steps: a) Evaluation of at least two different initial weights w _n (n = 1, 2,...) of the antioxidative substance and evaluation of the change of a signal intensity of the test radical I _wn (w _n = w ₁ , w ₂ , ...) (unit Arbitrary Units) for the at least two initial weights at least two different times t _m (m = 1 to 2 ...), b) normalization of the respective measured signal intensities I _wn (t) = I (t) to its respective output value I _wn (t = 0) = I ₀ , generation of a data set with n (n = 1, 2,...) Comparable normalized reduction curves by means of regression of the normalized signal intensities associated with the common weight w _n with a monoexponential function i _wn (t) and determination of the functional relationship i _wn (t) / I ₀ = f (t) according to

where I _0n corresponds to the signal intensity reached at the saturation point of the radical reduction for the respective weight w _n , and determination of the respective reaction _{constant of} the reduction process k _wn (w _n = w ₁ , w ₂ , w ₃ ,...) with the unit of measurement min ^-1 for the respective weight, and c) determination of the reaction time t _rn (unit min) according to the equation normalized to I ₀

Method according to Claim 2, characterized in that t _{rn is} generated by an automatic evaluation of the following functional relationship I _rn (t _rn ): I rn (t rn ) = I 0n + (1 - x) (I 0 - I 0n ) = I 0n + (I 0 - I 0n ) exp - (k wn · t rn ) (3) With (1 - x) = exp - (k wn · t rn ) (4). Method according to claim 2 or 3, characterized in that the factor x is an arbitrary number to be selected between 0 ... 1, which is constant for all weights w _n and describes the instant t _rn = t _r , at which the Signal intensity I ₀ by x times the total signal _decrease I ₀ - I _0n , of the total weight reduction caused by the weight w _n , has dropped. Method according to claim 4, characterized in that t _{r is} an averaging of all experimentally determined t _rn values according to

is. Method according to one of claims 2 to 5, characterized the factor x has a value of 1 / e = 0.368. Method according to one of the preceding claims, characterized in that the static parameter of the characteristic weight w _{c is determined} by means of the following, automatically proceeding steps: a) Arrangement of the signal amplitudes I _rn (t _rn ) / I determined for the time t _rn = t _{r 0} as a function of their assigned weights w = w _n (n = 1, 2, ...), generation of the functional relationship I _rn (t _rn ) / I ₀ = f (w _n ) and regression resulting from the functional relationship Functional curve by a mono-exponential function according to

b) Determination of the reaction constant k _tr (unit mg ^-1 ) from the mono-exponentially adjusted function curve, and c) determination of the characteristic weight w _c (unit mg / ml) as an amount which is necessary to reduce the maximum possible reducing power of x = (x * I ₀ ) / I ₀ by the factor x from I ₀ to I _c according to the equation

A method according to claim 7, characterized in that w _c by automatic evaluation of a function I _c (w _c ) according to I c (w c ) = (1 - x) I 0 + (I 0 - (1 - x) I 0 ) (1 - x) = (1 - x) I 0 + (I 0 - (1 - x) I 0 ) exp - (k tr · w c ) (9) With (1 - x) = exp - (k tr · w c ) (10) is determined. Method according to claim 7 or 8, characterized the factor x has the value 1 / e = 0.368. Method according to one of the preceding claims, characterized in that the parameter of the antioxidant power AP (unit [spins / mg · min] or [mol / l · min · mg] according to the equation

where RA is a radical radical characteristic of a test radical and N is the amount of free radicals thereby reduced, where N is determined by the number of free radical electron spins or by the concentration of the free radical of the test. A method according to claim 10, characterized in that RA by an automatic evaluation of a data set for determining the radical degradation according to RA = 1 - (1 - x 2 ) = x 2 (13) is produced. A method according to claim 10, characterized in that RA by an automatic evaluation of the functional relationship I _c (RA) according to RA = I 0 / I 0 - I c / I 0 (14) With I c = (1 - x) I 0 + (I 0 - (1 - x) I 0 ) · (1 - x) = I 0 - x 2 I 0 and I c / I 0 = 1 - x 2 (15) is produced. Method according to one of claims 10 to 12, characterized in that the radical degradation RA has a factor of x ² . A method according to claim 13, characterized in that the factor x ^{2 has} a value of (1 / e) ² = 0.135. Method according to one of the preceding claims, characterized marked that for The Antioxidant Power AP is a standard Antioxidant Unit (AU) is defined as being 1 AU of the Antioxidant Power AP solution of vitamin C at a concentration of 1 ppm. Method according to one of the preceding claims, characterized in that the measurement of the signal intensity I of the Testradikals performed by ESR spectroscopy. Method according to claim 16, characterized in that that as test radicals 2,2-diphenyl-1-picrylhydrazil (DPPH), 2,6-di-tert-butyl-alpha- (3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidenes) -p-tolyloxy (Galvinoxyl), Fremy's salt (solid salt of nitrosodisulfonic acid) and / or Nitroxyde be used. Method according to one of the preceding claims, characterized in that the measurement of the signal intensity I of the Testradikals done colorimetrisch in a UV / VIS spectrometer. Method according to claim 18, characterized that as a test radical 2,2-diphenyl-1-picrylhydrazil (DPPH) is used.